1) Regulation of retrograde mitochondrial transport in axons One of the most crucial organelles in axons are mitochondria. Mitochondria perform many functions important for local microenvironments including: 1) generate the energy necessary for cellular metabolism; 2) buffer calcium ion levels; and 3) supply ATP for the proper functioning of ion transporters that regulate neural excitability. In addition, the location of mitochondria has been shown to regulate axon branching. Not only do mitochondria need to be properly localized in axons to maintain axon health and function, mitochondria also need to move in order for them to maintain their own health: Mitochondria undergo fission-fusion dynamics which allow the exchange of proteins, lipids, and mitochondrial DNA. If these dynamics are disrupted, mitochondria rapidly undergo degradation. Consequently, mitochondrial transport is of the utmost importance for axon function and health. Our lab is working to identify the factors that regulate mitochondrial transport by the retrograde motor protein complex. Towards the end of my post-doctoral training, I discovered a mutant which lacks almost all retrograde mitochondrial movement. The causative mutation in this line results in depletion of Actr10 (actin related protein 10) a known component of the dynein-associated complex, dynactin. In vivo analyses of mitochondrial movement in actr10 mutants revealed a lack of retrograde mitochondrial movement but normal anterograde (non-dynein related) transport. Transport of other cargos assayed, including lysosomes and the dynein motor itself, was not altered in actr10 mutants. To determine if Actr10 was in fact necessary to link mitochondria to the retrograde motor, we performed mitochondrial fractionation experiments from actr10 mutants and wildtype siblings. These experiments confirmed that Actr10 is necessary for the dynein motor to interact with mitochondria. This linkage is likely not direct, however, as Actr10 does not have known membrane-associated domains. To identify the proteins which make up this link between Actr10 and mitochondria, we performed an immunoprecipitation experiment followed by mass spectrometry analysis. These experiments yielded a number of interesting candidates which we are currently testing for their role in retrograde mitochondrial transport in axons. Together, our work will define the mechanism of dynein-mitochondrial attachment for retrograde movement of this organelle in axons. 2) The function of retrograde mitochondrial motility in axons While we know quite a bit about the dynamics of mitochondrial motility in the short term (on the order of minutes), we know almost nothing about the life history of mitochondria in neurons over longer periods of time. Additionally, we do not understand the role of mitochondrial movement in neurons though it is clear that active transport of this organelle is critical to the health and function of neurons. To begin to address these long-standing questions in the field, we have generated tools to analyze mitochondrial localization, transport, health, and function in vivo in zebrafish. Additionally, we have established collaborations to analyze neural circuit function when mitochondrial transport is inhibited. Together, this analysis revealed a critical role for mitochondrial retrograde transport in the maintenance of a homeostatic mitochondrial distribution in neurons. Inhibiting mitochondrial retrograde flux resulted in a depletion of cell body mitochondria with organelle accumulation in the distal axon. Accumulated mitochondria show signs of failed health and can no longer buffer calcium. This leads to impaired neural circuit activity. While informative, this left an obvious question of why mitochondria need to move back to the cell body. We reasoned that mitochondria could be moved back to this compartment for protein replenishment. Mitochondria have >1200 proteins, some of which turnover on the order of hours. 99% of these proteins are encoded by nuclear DNA. Perhaps it would make more sense to move the organelle back to the region of protein synthesis rather than bring the proteins to the organelle. Using mass spec analysis of the mitochondrial proteome, we revealed an essential role for retrograde mitochondrial transport for maintenance of the mitochondrial proteome. Disrupting this process leads to a >50% loss of almost a hundred mitochondrial proteins. Together, our work defines the dynamics of mitochondrial transport in neurons and has shown for the first time that retrograde movement specifically is required for mitochondrial protein replenishment and organelle homeostasis in neurons. 2) Identifying novel regulators of retrograde cargo transport in axons Forward genetics is an ideal and unbiased way to identify proteins with critical functions in cellular processes. We have initiated a forward genetic screen in zebrafish to identify proteins important for the retrograde transport of specific cargos in axons. For this screen, we are using a transgenic line that marks both the sensory and motor neuron axons in zebrafish with cytoplasmic GFP (Green Fluorescent Protein). Because cargos that fail to undergo retrograde transport accumulate over time in axon terminals, we can screen our mutagenized families for axon terminal size using the GFP fluorescent indicator to identify strains with disruptions in retrograde axonal transport. In addition to being an efficient screening procedure, the ability to screen live at various developmental stages also gives us the flexibility necessary to study multiple types of axons that develop at different time-points in the same animals. Additionally, our transgenic line contains a second transgene to label mitochondria with the red fluorescent protein TagRFP. Consequently, our screen will also allow us to identify mutant strains with defect in mitochondrial positioning as well as more general markers of retrograde transport disruption. Together, this screen will identify novel regulators of retrograde cargo transport and regulators of mitochondrial localization and motility in axons. This will advance our goal of defining the mechanisms of cargo-specific retrograde transport in axons.
| Herbert, Amy L; Fu, Meng-Meng; Drerup, Catherine M et al. (2017) Dynein/dynactin is necessary for anterograde transport of Mbp mRNA in oligodendrocytes and for myelination in vivo. Proc Natl Acad Sci U S A 114:E9153-E9162 |
| Drerup, Catherine M; Herbert, Amy L; Monk, Kelly R et al. (2017) Regulation of mitochondria-dynactin interaction and mitochondrial retrograde transport in axons. Elife 6: |
